Methods for identifying enzyme inhibitors

ABSTRACT

A method for identifying an inhibitor of dehydroquinate synthase (DHQS) and/or dehydroquinase (DQ), which method comprises: i) contacting a test substance with DHQS and a substrate for DHQS and contacting the resulting reaction mixture with DQ, or contacting the test substance with DQ and a substrate for DQ; and ii) contacting the resulting reaction mixture with dehydroshikimate dehydratase (DHSD); and iii) determining whether the test substance inhibits the activity of DHQS or DQ.

FIELD OF THE INVENTION

This invention relates to methods for identifying substances capable ofinhibiting the enzymes dehydroquinate synthase (DHQS) and/ordehydroquinase (DQ). It further relates to DHQS and/or DQ assays foridentifying activity in a sample and to test kits for identifyingsubstances capable of inhibiting dehydroquinate synthase and/ordehydroquinase.

BACKGROUND TO THE INVENTION

The shikimate pathway is an ancient pathway that is involved in primaryand secondary metabolism and is found in all prokaryotes, many lowereukaryotes and plants, but not in mammalian cells. In primary metabolismthe function of the pathway is to provide the precursors for theproduction of the aromatic amino acids and para-aminobenzoic acid. Theshikimate pathway includes the enzymes and metabolites formed byconverting 3-deoxy D-arabino-heptulosonic 3-phosphate (DAHP) tochorismic acid, the trifurication point for the three pathways leadingto the production of tryptophane, tyrosine and phenylalanine.

In some microbial eukaryotes and prokaryotes, two of the products of theshikimate pathway (dehydroquinate and dehydroshikimate) are also sharedby the quinate utilisation (qut) pathway (Hawkins et al., Molec. Gen.Genet. 214, 224-231, 1988). The qut pathway is a dispensable carbonutilisation pathway and the application of metabolic control analysishas shown that the common intermediates approximate an open pool and canbe fluxed within and between the shikimate and qut pathways (Lamb etal., Molec. Gen. Genet. 227, 187-196, 1991; Lamb et al., Biochem. J.284, 181-187, 1992 and Wheeler et al., Biochem. J. 315, 195-205, 1996).The biochemical relationships between the shikimate and quinate pathwaysare summarised in FIG. 1.

Overproduction of the gut pathway enzyme dehydroshikimate dehydratase inthe absence of quinate causes an auxotrophic requirement for thearomatic amino acids due to flux of shikimate pathway dehyroshikimate tothe gut pathway end point protocatechuic acid (Lamb et al., 1992). InAspergillus nidulans for example it is advantageous to the growingmycelium that the qut pathway enzymes are only produced when quinate isavailable as a carbon source as their production in its absence woulddeplete flux in the essential shikimate pathway.

In A.nidulans the gut pathway is controlled by two transcriptionregulating proteins (designated QUTA and QUTR) that interact to ensurethat the qut enzymes are only present when quinate is available (Beri etal., Nucleic Acids Res. 19, 7991-8001, 1987; Hawkins et al., Gene 110,109-114, 1992; Hawkins et al., Gene 136, 49-54, 1993).

The importance of the shikimate pathway to cell viability is illustratedby experiments that result in the disruption of enzyme function. Inplants, the shikimate pathway enzyme EPSP synthase has been targeted bya chemical inhibitor strategy that has resulted in the commerciallysuccessful broad range post-emergent herbicide called glyphosate.

In various microbial species, analysis of the shikimate pathway has beencarried out genetically by the construction of mutants. When mutants ofvirulent prokaryotic or microbial eukaryotic species lacking enzymes atvarious steps in this pathway, the so-called aro⁻ mutants, are used toinfect animals, their virulence is generally observed to be attenuated(Leech et al., J. Biol. Chem. 270, 25827-25836, 1995 and Gunel-Ozcan etal., Microbial Pathogen. 17, 169-174, 1997). After infection with aro⁻mutants of S.typhimurium, mice are resistant to further challenge withthe wild type strain. The probable reason for attenuation andimmunological protection is that these aro⁻ mutants strains persist inthe host-and replicate at a greatly reduced rate, thereby stimulatingcell mediated immunity. The reason that aro⁻ mutants strains persistbefore being cleared is probably because they are able to derivesufficient quantities of the aromatic amino acids from the host cells toprevent immediate death. However, it is likely that their growth islimited by the availability of para-aminobenzoic acid.

Recently, the shikimate pathway has been characterised in apicomplexanparasites such as Toxoplasma gondii, Plasmodium falciparum (malaria) andCryptosporidium parvum (Roberts et al., Nature 393, 801-805, 1998).Importantly, the growth of these parasites can be inhibited by theherbicide glyphosate, suggesting that the shikimate pathway will make agood target for the development of new anti-parasite agents.

The observations that both chemical and genetic inhibition of theshikimate pathway results in reduced cell viability has stimulatedinterest in the pathway as a possible target for drug therapy in acutemicrobial infection. It is likely that compounds which can inhibit theactivity of shikimate enzymes will not cause cell death of the infectingmicrobe, but will result in attenuation in a manner analagous to thephenotype of shikimate pathway mutants. As antimicrobials, thesecompounds may be expected to induce stasis rather than cell lysis ordeath, allowing the infection to be cleared by the host's immune system.Such an outcome is desirable as it will ameliorate the absoluteselective pressure to select for the growth of resistant mutants whichwould inevitably be the case if the compounds used caused cell death.Additionally this strategy may also result in a degree of immuneprotection which may prevent reinfection. As efficacious compounds areunlikely to kill any infecting microorganisms, then the risks of toxicshock caused by, for example, bacterial protein and cellular debris willbe minimised when treatment is administered.

SUMMARY OF THE INVENTION

According to the present invention there is provided a method foridentifying an inhibitor of dehydroquinate synthase (DHQS) and/ordehydroquinase (DQ) comprising:

(i) contacting a test substance with DHQS and a substrate for DHQS andcontacting the resulting reaction mixture with DQ, or contacting thetest substance with DQ and a substrate for DQ; and

(ii) contacting the resulting reaction mixture with dehydroshikimatedehydratase (DHSD); and

(iii) determining whether the test substance inhibits the activity ofDHQS or DQ.

The invention also provides:

a method of identifying DHQS activity in a sample, comprising:

(i) contacting the sample with a substrate for DHQS and contacting theresulting reaction mixture with DQ;

(ii) contacting the resulting reaction mixture with DHSD; and

(iii) determining whether the sample exhibits DHQS activity;

a method of identifying DQ activity in a sample, comprising:

(i) contacting the sample with a substrate for DQ;

(ii) contacting the resulting reaction mixture with DHSD; and

(iii) determining whether the sample exhibits DQ activity;

a test kit suitable for use in identifying an inhibitor of DHQS, whichkit comprises DHQS, a substrate for DHQS, DQ, DHSD and a buffer; and

a test kit suitable for use in identifying an inhibitor of DQ, which kitcomprises DQ, a substrate for DQ, DHSD and a buffer.

The invention thus provides flexible assays for dehydroquinate synthase(DHQS) and dehydroquinase (DQ). These assays couple the production ofdehydroquinate (produced by dehydroquinate synthase) to either a type Ior a type II dehydroquinase (which convert dehydroquinate todehydroshikimate) and a dehydroshikimate dehydratase (which convertsdehydroshikimate to protocatechuic acid). The product dehydroshikimatecan be monitored at 237 nm, and/or the product protocatechuate can bemonitored at either 290 nM or, after reaction with iron, at 547 nM. Thismeans that the activity of the enzyme DHQS or DQ can be measuredcontinuously at two different points in the uv spectrum or by adiscontinuous assay in the visible spectrum.

The assay for DHQS and DQ can be used to identify inhibitors of DHQS andDQ. The fact that the assay for inhibitors can be carried out as adiscontinuous assay in the visible spectrum has the advantage that cheapplastic microtitre plates can be used to detect the effects of specificcompounds on DHQS and/or DQ activity. Also, the screen for inhibitorsubstances is not limited by the absorbance spectrum of the substancebeing tested because the activity of DHQS and DQ can be measured atdifferent points in the uv and/or visible spectra. The assay is suitablefor adaptation to 96 well and 384 well plate technologies and can beautomated using liquid handling robots, allowing modern high throughputscreening techniques to be applied. The invention therefore permits highthrough-put, flexible and inexpensive screening for substances whichinhibit DHQS and DQ. This will increase the likelihood of potentbioavailable drugs being identified.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the biochemical relationships between the shikimate andquinate pathways; and

FIG. 2a shows the results obtained using the enzyme and substratemixtures described in Table 1 below. The rows are numbered 1 to 6according to mixtures 1 to 6 of Table 1.

FIG. 2b shows the results obtained using the enzyme and substratemixtures described in Table 2 below. The rows are numbered 1 to 6according to mixtures 1 to 6 of Table 2.

DETAILED DESCRIPTION OF THE INVENTION

Enzymes

Any DHQS, DQ and DHSD may be used. The enzymes may be prokaryotic oreukaryotic. They may be obtained from prokaryotic or eukaryoticextracts, for example from a microbial extract. Alternatively, theenzymes may be produced recombinantly, from, for example, bacteria,yeast or higher eukaryotic cells such as insect cell lines.

(i) Dehydroquinate Synthase (DHQS)

The enzyme dehydroquinate synthase catalyses the second step in theshikimate pathway and is an ideal candidate as a target forchemotherapeutic design. The DHQS substrate is typically 3-deoxyD-arabino-heptulosonic 3-phosphate (DAHP). NAD is typically also presentas a catalytic substrate for the enzymatic reaction.

A preferred enzyme is the DHQS from A.nidulans. The reason for thischoice is that its reaction mechanism has been studied and characterisedin great detail and recently the structure of this enzyme has beeneludicated to a resolution of 1.8 angstroms. The N-terminal domain ofthe AROM protein from A.nidulans corresponding to the DHQS enzyme hasbeen crystallised with an active site inhibitor and with one of thesubstrates NAD⁺. Based on the crystal structure, key amino residues havebeen identified and their involvement in the reaction mechanismconfirmed by site-directed mutagenesis. Large quantities of thewild-type enzyme can be purified with ease and mutant forms lackingenzyme activity can similarly be purified in bulk.

(ii) Dehydroquinase (DQ)

A type I or type II DQ may be used. Preferred examples of DQ are the DQenzyme from Salmonella typhi and Mycobacterium tuberculosis. The crystalstructures of the type I DQ from Salmonella typhi has been determined to2.1 angstroms and the type II DQ from Mycobacterium tuberculosis to 2angstroms. The details of the reaction mechanisms for each protein areknown and the role of some of the amino acids implicated in the reactionmechanism have been investigated by site-directed mutagenesis.

Both of these enzymes can be purified in bulk and either enzyme can beused to catalyse the product of the dehydroquinate synthase enzyme,dehydroquinate to dehydroshikimate the substrate for dehydroshikimatedehydratase. The DQ substrate is thus dehydroquinic acid(dehydroquinate).

(iii) Dehydroshikimate Dehydratase (DHSD)

A preferred enzyme is the A.nidulans DHSD.

Assays

Any suitable format may be used for the assay for identifying aninhibitor of DHQS and/or DQ. The DHSD may be added to the mixtureresulting from step (i). More usually, however, steps (i) and (ii) areconducted as a single step. Thus, the test substance may be contactedwith DHQS, a substrate for DHQS, DQ and DHSD when testing for aninhibitor of DHQS. The test substance may be contacted with DQ, asubstrate for DQ and DHSD when testing for a DQ inhibitor. The assay isgenerally therefore carried out in a single medium. Most preferably theassay is carried out in a single well of a plastic microtitre plate.

In practice, the enzyme reactions are commenced by addition of DHQS or asubstrate for DHQS or, when testing for a DQ inhibitor, DQ or asubstrate for DQ. An assay for a DHQS inhibitor may therefore beinitiated by providing a medium, typically a buffered medium, containinga test substance and one of DHQS and a DHQS substrate, and adding theother of DHQS and the DHQS substrate to the medium. The medium may alsocontain DQ and DHSD. An assay for a DQ inhibitor may be initiated byproviding a medium, such as a buffered medium, containing a testsubstance, one of DQ and a DQ substrate and, optionally, DHSD, andadding the other of DQ and the DQ substrate to the medium.

However, the assay for a DHQS inhibitor may be carried out by thesequential contact of DHQS, DQ and DHSD with the substance to be tested.In such an assay the substance to be tested would be contacted witheither DHQS or a substrate for DHQS and the reaction initiated by theaddition of a substrate for DHQS or DHQS respectively. DQ is then addedto the reaction mixture or an aliquot could be removed from the reactionmixture and contacted with DQ in a separate mixture. Subsequently, DHSDcan be added to the resulting reaction mixture or an aliquot could beremoved from the reaction mixture and contacted with DHSD in a separatemixture. A sequential assay for a DQ inhibitor may be performedsimilarly.

The course of an assay can be followed by monitoring absorbance at 237nm. Dehydroshikimate (dehydroshikimic acid) production can be monitoredat this wavelength. The course of the assay may additionally oralternatively be followed by monitoring absorbance at 290 nm. Theaccumulation of the final product protocatechuic acid (PCA) can bemonitored at this wavelength. Alternatively, the reaction can beterminated by adding a soluble ferric salt such as ferric chloride, forexample FeCl₃.6H₂O. The PCA reacts with the iron to form a complex whichcan be measured in the visible spectrum at 547 nm.

The assay can thus be followed by measuring the change in absorbance ofthe assay medium due to accumulation of the final product,protocatechuic acid (PCA).

The assay of the invention may be carried out at any temperature atwhich DHQS, DQ and DHSD, in the absence of any inhibitor are active.Typically, however, the assay will be carried out in the range of from25° C. to 37° C.

The assay is typically carried out in a reaction buffer comprising NAD,a source of Zn²⁺ ions and a source of Mg²⁺ ions. The buffer may be aBisTrisPropane buffer. Preferably the buffer is 12.5 mMBisTrisPropane/acetate pH 7.0, 40 μM ZnSO₄ and 2.5 mM MgSO₄. The buffercan also contain 125 μM NAD for assay at 290 nm and 250 μM NAD for assayat 547 nm. An assay mixture could therefore be made up consisting of:

12.5 mM BisTrisPropane/acetate pH 7.0;

40 μM ZnSO₄;

2.5 mM MgSO₄;

125 μM NAD if the assay is to be monitored at 290 nm or 250 μM NAD ifthe assay is to be monitored at 547 nm;

40 μM DAHP if the assay is to be monitored at 290 nm or 286 μM DAHP ifthe assay is to be monitored at 547 nm;

1.0 units per ml of the Salmonella typhi type I DQ; and

1.0 units per ml of the A.nidulans DHSD.

An assay utilising such an assay mixture would typically be initiated bythe addition of A.nidulans DHQS to a concentration of 0.1 units per ml.

As a control, the progress of the assay can be followed in the absenceof the substance to be tested. Further control experiments can becarried out. For example, the ability of the substance being tested toinhibit the activity of DQ as well as DHQS can be identified bycontacting the said substance with DQ and a substrate for DQ. Theresulting reaction mixture can be contacted with DHSD, to determine theability of the substance to inhibit the activity of DQ. Such a controlwill allow the skilled man to determine whether a substance inhibitsDHQS or DQ or indeed both.

Additionally, a substance to be tested could be contacted with DHSD anda substrate for DHSD to determine the ability of the substance toinhibit the activity of DHSD.

Test Substances

A substance which inhibits the activity of DHQS and/or DQ may do so bybinding to one or both of the enzymes. Such enzyme inhibition may bereversible or irreversible. An irreversible inhibitor dissociates veryslowly from its target enzyme because it becomes very tightly bound tothe enzyme, either covalently or non-covalently. Reversible inhibition,in contrast with irreversible inhibition, is characterised by a rapiddissociation of the enzyme-inhibitor complex.

The test substance may be a competitive inhibitor. In competitiveinhibition, the enzyme can bind substrate (forming an enzyme-substratecomplex) or inhibitor (enzyme-inhibitor complex) but not both. Manycompetitive inhibitors resemble the substrate and bind the active siteof the enzyme. The substrate is therefore prevented from binding to thesame active site. A competitive inhibitor diminishes the rate ofcatalysis by reducing the proportion of enzyme molecules bound to asubstrate.

The inhibitor may also be a non-competitive inhibitor. Innon-competitive inhibition, which is also reversible, the inhibitor andsubstrate can bind simultaneously to an enzyme molecule. This means thattheir binding sites do not overlap. A non-competitive inhibitor acts bydecreasing the turnover number of an enzyme rather than by diminishingthe proportion of enzyme molecules that are bound to substrate.

The inhibitor can also be a mixed inhibitor. Mixed inhibition occurswhen an inhibitor both effects the binding of substrate and alters theturnover number of the enzyme.

A substance which inhibits the activity of DHQS or DQ may also do so bybinding to the substrate. The substance may itself catalyze a reactionof the substrate, so that the substrate is not available to the enzyme.Alternatively the inhibitor may simply prevent the substrate binding tothe enzyme.

Suitable candidate substances include antibody products (for example,monoclonal and polyclonal antibodies, single chain antibodies, chimaericantibodies and CDR-grafted antibodies) which are specific for DHQSand/or DQ. Furthermore, combinatorial libraries, defined chemicalidentities, peptide and peptide mimetics, oligonucleotides and naturalproduct libraries may be screened for activity as inhibitors of DHQSand/or DQ in assays such as those described below. The candidatesubstances may be used in an initial screen of, for example, tensubstances per reaction, and the substance of these batches which showinhibition tested individually. Candidate substances which show activityin assays such as those described below can then be tested in in vivosystems, such as an animal model. Candidate inhibitors could be testedfor their ability to attenuate microbial infection in mice.

Therapeutic Uses

Virulent prokaryotic or microbial eukaryotic species mutant for enzymesat various steps of the shikimate pathway are generally observed to beattenuated when used to infect animals. Furthermore, the apicomplexanparasites have been observed to be inhibited by the herbicideglyphosphate. The present invention can enable a substance to beidentified which is capable of inhibiting the activity of one or both oftwo enzymes of the shikimate pathway, DHQS and DQ. In particular, such asubstance may be used in a method of treating a microbial, especiallybacterial, infection. Such substances may also be used for themanufacture of a medicament for use in the treatment of a microbialinfection.

The formulation of a substance identified according to the inventionwill depend upon the nature of the substance identified. Typically asubstance is formulated for clinical use with a pharmaceuticallyacceptable carrier or diluent. For example it may be formulated fortypical parenteral, intravenous, intramuscular, subcutaneous,intraocular, transdermal or oral administration. A physician will beable to determine the required route of administration for a particularpatient and the condition. The pharmaceutical carrier or diluent may be,for example, an isotonic solution.

The dose of substance used may be determined according to variousparameters, especially according to the substance used; the age, weightand condition of the patient to be treated; the route of administration;and the required clinical regimen. A physician will be able to determinethe required route of administration and dosage for any particularpatient and condition.

Test Kits

The test kits of the invention comprise DQ and DHSD. When the test kitis intended for use in assaying for a DHQS inhibitor, the kit alsocontains DHQS and a substrate for DHQS. When the test kit is intendedfor use in assaying for a DQ inhibitor, the kit contains DQ and a DQsubstrate.

The kits also contain a buffer. Typically the buffer comprises NAD, asource of Zn²⁺ ions and a source of Mg²⁺ ions. Preferably the buffer is12.5mM BisTrisPropane/acetate pH 7.0, 40 μM ZnSO₄ and 2.5 mM MgSO₄. Thebuffer may also contain 125 μM NAD for assays which are to be monitoredat 290 nm and 250 μM NAD for assays which are to be monitored at 547 nm.An soluble ferric salt may be provided, such as ferric chloride, whichcan be added to the PCA product formed as a result of the coupled enzymereaction. The ferric chloride is typically in the form FeCl₃.6H₂O.

The following Example illustrates the invention.

EXAMPLE

Abbreviations

AN-DHQS Aspergillus nidulans dehydroquinate synthase.

STI Salmonella typhi type I dehydroquinase.

MTII Mycobacterium tuberculosis type II dehydroquinase.

ANII Aspergillus nidulans type II dehydroquinase.

AN-DHSD Aspergillus nidulans dehydroshikimate dehydratase.

DAHP 3-deoxy D-arabino-heptulosonic 3-phosphate.

NAD Nicotinamide Adenine Di-Nucleotide.

Materials

DAHP was purified according to the method of Shujaath et al., Methods inEnzymol. 142, 306-314, 1987. The AN-DHQS, STI, ANII, MTII and AN-DHSDwere purified according to the protocols described in references Mooreet al., Biochem. J. 301, 297-304, 1994; Moore et al., Biochem J. 295,277-285, 1993; Gourley et al., J. Mol. Biol. 241, 488-491, 1994; andWheeler et al., Biochem J. 315, 195-205, 1996. The buffer was 12.5 mMBisTrisPropane/acetate pH 7.0 containing 40 μM ZnSO₄ and 2.5 mM MgSO₄.

Methods

NAD, AN-DHQS, ANII/MTII and AN-DHSD enzymes were added to 200 μlaliquots of the buffer and thoroughly mixed by gentle pipetting. Thesubstrate DAHP was pipetted directly into the wells of a microtitreplate immediately before initiating the reaction. The reactions wereinitiated by the addition of 50 μl of the enzyme/NAD mixture to themicrotitre wells containing the substrate DAHP. The contents of themicrotitre wells were mixed by being drawn up and expelled five timeswith a disposable plastic tip designed for use on a 200 μl adjustablepipette.

The microtitre plate was then transferred to a dry incubator set at 37°C. and incubated for 15 minutes. The microtitre plate was then removedfrom the incubator and 1 μl of a 1% (w/v) solution of FeCl₃.6H₂O wasadded to each of the reaction mixtures. Addition of the iron to thosemixtures containing all three enzymes caused an intense localisedblue/black colour to appear. The individual reaction mixtures were mixedby drawing up and expelling each mixture five times with a disposableplastic tip designed for use on a 200 μl adjustable pipette. Thisresulted in the appearance of an even blue colour in solution which wasthen photographed.

Results

1. Control experiments were carried out to demonstrate that the bluecolour formed by the complex of iron with protocatechuate isspecifically formed due to the sequential action of the enzymesdehydroquinate synthase, dehydroquinase and dehydroshikimatedehydratase. The assays were carried out in triplicate using the fullcomplement of three enzymes. These assays used the S.typhi type Idehydroquinase or the type II enzymes from M. tuberculosis or A.nidulansas alternate sources of the first linking enzyme. The assays wererepeated in triplicate leaving out singly each of the three enzymes. Theprecise enzyme and substrate mixtures are shown in Table 1 below:

Buffer AN- AN- (without 2.5 mM 12 mM DHQS STI MTII ANII DHSD NAD) NADDAHP 1 0.02 U 0.2 U 0    0    0.16 U 200 μl 2 μl 2.5 μl 2 0.02 U 0   0.2 U 0    0.16 U 200 μl 2 μl 2.5 μl 3 0.02 U 0    0    0.2 U 0.16 U 200μl 2 μl 2.5 μl 4 0.02 U 0    0    0    0.16 U 200 μl 2 μl 2.5 μl 5 0.02U 0    0    0.2 U 0    200 μl 2 μl 2.5 μl 6 0    0    0    0.2 U 0.16 U200 μl 2 μl 2.5 μl

FIG. 2a shows the result. If any one of the three enzymes is left out noblue colouration was produced, demonstrating that its formation wasenzyme-dependent.

2. The full assay (using the A.nidulans enzyme) was repeated intriplicate with a two-fold serial dilution of the substrate DAHP in therange 286 μM to 9 μM. This is shown in Table 2 below:

AN- AN- 2.5 mM 12 mM Final DAHP DHQS ANII DHSD Buffer NAD DAHPconcentration 1 0.02 U 0.2 U 0.16 U 200 μl 2 μl 5 μl 286 (μM) 2 0.02 U0.2 U 0.16 U 200 μl 2 μl 2.5 μl 145 (μM) 3 0.02 U 0.2 U 0.16 U 200 μl 2μl 2.5 μl of  72 (μM) 1 in 2 dilution 4 0.02 U 0.2 U 0.16 U 200 μl 2 μl2.5 μl of  36 (μM) 1 in 4 dilution 5 0.02 U 0.2 U 0.16 U 200 μl 2 μl 2.5μl of  18 (μM) 1 in 8 dilution 6 0.02 U 0.2 U 0.16 U 200 μl 2 μl 2.5 μlof  9 (μM) 1 in 16 dilution

The results are shown in FIG. 2b.

Discussion

These experiments show that the extent of blue colouration (productformation) is dependent on substrate concentration. Taken together thecombined assays show that blue colouration is enzyme- andsubstrate-dependent. This means that the substrate concentration can bemodulated to produce colour intensities that fall within the linearmeasuring range of the microtitre plate reader. Furthermore, the Exampledemonstrates that if any one of the enzymes is inhibited (eithercompletely or partially) by a chemical addition to the assay then thiswould be easily detectable by an automated plate reader.

What is claimed is:
 1. A method for identifying an inhibitor ofdehydroquinate synthase (DHQS) and/or dehydroquinase (DQ), which methodcomprises: (i) contacting a test substance with DHQS and a substrate forDHQS and contacting the resulting reaction mixture with DQ, orcontacting the test substance with DQ and a substrate for DQ; and (ii)contacting the resulting reaction mixture with dehydroshikimatedehydratase (DHSD); and (iii) determining whether the test substanceinhibits the activity of DHQS or DQ by monitoring the amount ofdehydroshikimate and/or protocatechuate by absorbance.
 2. A methodaccording to claim 1, wherein a buffered medium containing the testsubstance, DQ, DHSD and DHQS is provided and the enzyme reaction is theninitiated by adding the DHQS substrate to the said buffered medium.
 3. Amethod according to claim 1, wherein the DHQS is Aspergillus nidulansDHQS and/or the substrate for DHQS is 3-deoxy D-arabino-heptulosonic3-phosphate (DAHP).
 4. A method according to claim 1, wherein a bufferedmedium containing the test substance, DHSD and DQ is provided and theenzyme reaction is then initiated by adding the DQ substrate to the saidbuffered medium.
 5. A method according to claim 1, wherein the DQsubstrate is dehydroquinic acid.
 6. A method according to claim 1,wherein the DQ is selected from the group consisting of type Idehydroquinase from Salmonella typhi, type II dehydroquinase fromMycobacterium tuberculosis and type II dehydroquinase from Aspergillusnidulans.
 7. A method according to claim 1, wherein the DHSD isAspergillus nidulans DHSD.
 8. A method according to claim 1, whereinabsorbance of dehydroshikimate is measured at 237 nm and/or absorbanceof protocatechuate is measured at 290 nm.
 9. A method according to claim1, wherein the reaction mixture resulting from step (ii) is contactedwith a soluble ferric salt.
 10. A method according to claim 9, whereinabsorbance of the reaction mixture in which the soluble ferric salt hasbeen provided is measured at 547 nm.
 11. A method of identifying DHQSactivity in a sample, which method comprises: (i) contacting the samplewith a substrate for DHQS and contacting the resulting reaction mixturewith DQ; (ii) contacting the resulting reaction mixture with DHSD; and(iii) determining whether the sample exhibits DHQS activity bymonitoring the amount of dehydroshikimate and/or protocatechuate byabsorbance.
 12. A method of identifying DQ activity in a sample, whichmethod comprises: (i) contacting the sample with a substrate for DQ;(ii) contacting the resulting reaction mixture with DHSD; and (iii)determining whether the sample exhibits DQ activity by monitoring theamount of dehydroshikimate and/or protocatechuate by absorbance.
 13. Atest kit suitable for use in identifying an inhibitor of DHQS or DO,which kit comprises DQ, DHSD, a buffer and (a) DHQS and a substrate forDHQS or (b) a substrate for DQ.
 14. A method according to claim 1,wherein a buffered medium containing the test substance, DQ, DHSD andthe DHQS substrate is provided and the enzyme reaction is then initiatedby adding DHQS to the said buffered medium.
 15. A method according toclaim 1, wherein a buffered medium containing the test substance, DHSDand the DQ substrate is provided and the enzyme reaction is theninitiated by adding DQ to the said buffered medium.